Nikki J. Marks

Parasitic Flatworms: Molecular Biology, Biochemistry, Immunology and Physiology

Part I: Phylogeny, Genetics and Transcriptomes * The Evolution of Parasitism in Flatworms, D T J Littlewood, Natural History Museum, UK * Genomes and Genomics of Parasitic Flatworms, D A Johnston, Natural History Museum, UK * Genetic Discrimination of Echinococcus Species and Strains, D P McManus, University of Queensland, Australia * Ribosomal DNA Variation in Parasitic Flatworms, D Blair, James Cook University, Australia * Genetic Studies on Monogeneas with Emphasis on Gyrodactylus, C O Cunningham and I Matejusovo, FRS Marine Laboratory, UK * The Schistosome Transcriptome, S Verjovski-Almeida and R DeMarco, Universidade de Sao Paulo, Brazil * Transgenic Flatworms, C G Grevelding, Justus-Liebig-University Giessen, Germany Part II: Immunobiology, Host-Parasite Interaction and Control * Immunobiology of Schistosomes, S G Forrester, University of Ontario, Canada and E J Pearce, University of Pennsylvania, USA * Cestode Infection: Immunological Considerations From Host and Tapeworm Perspectives, D M McKay and R A Webb, York University, Canada * Parasitic Flatworms: Signal Transduction at the Host-Parasite Interface, T P Yoshino, J J Vermeire and J Humphries, University of Wisconsin-Madison, USA * Parasite Effects on the Snail Host Transcriptome, M Knight and N Raghavan, Biomedical Research Institute, USA * Developments in the chemotherapy of parasitic flatworms, G C Coles, University of Bristol, UK * Drug Resistance in Schistosomes, T A Day, Iowa State University, USA and S Botros, Theodor Bilharz Research Institute, Egypt * Praziquantel: Mechanism of Action, R M Greenberg, Marine Biological Laboratory, USA * Cestode Vaccine Development, M W Lightowlers, University of Melbourne, Australia * The Development of a Schistosome Vaccine, R A Wilson and P S Coulson, University of York, UK Part III: Protein Function, Metabolism and Physiology * Flatworm Parasite Proteomics, R M Morphew, J Barrett and P M Brophy, University of Wales, UK * Proteases in Trematode Biology, J P Dalton, University of Technology, Australia, C R Caffrey, M Sajid, University of California, USA, C Stack, S Donnelly, University of Technology, Australia, A Loukas, T Don, Queensland Institute of Medical Research, J McKerrow, University of California, USA, D W Halton, Queens University Belfast, UK and P J Brindley, Tulane University Health Sciences Center, USA * Signalling Molecules and Nerve-Muscle Function, A G Maule, N J Marks and T A Day * Unusual Aspects of Metabolism in Flatworm Parasites, A G M Tielens and J J van Hellemond, Utrecht University, Netherlands * Glycoconjugate Structures, M Wuhrer, Leiden University Medical Center, Netherlands and R Geyer, University of Giessen, Germany * Gene Silencing in Flatworms using RNA interference, P J Skelly, Tufts University School of Veterinary Medicine, USA.

RNAi effector diversity in nematodes

While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes.

Inhibitory effects of nematode FMRFamide-related peptides (FaRPs) on muscle strips fromAscaris suum

A large number of FMRFamide-related peptides (FaRPs) are found in nematodes, and some of these are known to influence tension and contractility of neuromuscular strips isolated fromAscaris suum body wall. Relaxation of these strips has been noted with five nematode FaRPs. The inhibitory actions of SDPNFLRFamide (PF1) and SADPNFLRFamide (PF2) appear to be mediated by nitric oxide, as previously demonstrated with inhibitors of nitric oxide synthase (NOS). This present study showed that the effects of PF1 were also dependent on external Ca++ and were reduced by the Ca++-channel blocker verapamil, observations consistent wirh the finding that nematode NOS is Ca++-dependent. KSAYMRFamide (PF3), KPNFIRFamide (PF4) and KNAFIRFamide (an alanine substituted analog of KNEFIRFamide, AF1, termed A3AF1) also relaxed A.suum muscle strips, but these responses were not affected by NOS inhibitors. PF3 inhibited the activity of strips prepared from the dorsal side of the worm, but contracted ventral strips. Both effects were dependent on the presence of ventral/dorsal nerve cords (unlike PF1/PF2) and were attenuated in medium which contained high K+ or low Ca++. PF4-induced muscle relaxation and hyperpolarization were independent of nerve cords, but were reversed in Cl-free medium, unlike PF1 or PF3. The PF4 effect physiologically desensitized muscle strips to subsequent treatment with PF4 and/or GABA. However, PF4 and GABA were not synergistic in this preparation. The effects of GABA, but not PF4, were reduced in muscle strips treated with the GABA antagonist, NCS 281-93. Following PF4 (or GABA) relaxation, subsequent treatment with higher doses of PF4 caused muscle strip contraction. A3AF1 was found to relax muscle strips and hyperpolarize muscle cells independently of the ventral and dorsal nerve cords, K+, Ca++, and Cl-, and mimicked the inhibitory phase associated with the exposure of these strips to AF1. On the basis of anatomical and ionic dependence, these data have delineated at least four distinct inhibitory activities attributable to nematode FaRPs. Clearly, a remarkably complex set of inhibitory mechanisms operate in the nematode neuromuscular system.

Neuropeptide Signaling Systems - Potential Drug Targets for Parasite and Pest Control.

Current problems of drug resistance in parasites and pests demand the identification of new targets and their exploitation through novel drug design and development programs. Neuropeptide signaling systems in helminths (nematodes and platyhelminths = worms) and arthropods are well developed and complex, play a crucial role in many aspects of their biology, and appear to have significant potential as targets for novel drugs. The best-known neuropeptide family in invertebrates is the FMRFamide-related peptides (FaRPs). Amongst many roles, FaRPs potently influence motor function. The genome sequencing projects of Drosophila melanogaster and Caenorhabditis elegans have revealed unexpected complexity within the FaRPergic systems of arthropods and nematodes, although available evidence for platyhelminths indicates structural and functional simplicity. Regardless of these differences, FaRPs potently modulate motor function in arthropods, nematodes and platyhelminths and there appears to be at least some commonality in the FaRPergic signaling systems therein. Moreover, there is now increasing evidence of cross-phyla activity for individual FaRPs, providing clear signals of opportunities for target selection and the identification and development of broad-spectrum drugs.

Pharmacology of FMRFamide-related Peptides in Helminths

Abstract: Nervous systems of helminths are highly peptidergic. Species in the phylum Nematoda (roundworms) possess at least 50 FMRFamide‐related peptides (FaRPs), with more yet to be identified. To date, few non‐FaRP neuropeptides have been identified in these organisms, though evidence suggests that other families are present. FaRPergic systems have important functions in nematode neuromuscular control. In contrast, species in the phylum Platyhelminthes (flatworms) apparently utilize fewer FaRPs than do nematodes; those species examined possess one or two FaRPs. Other neuropeptides, such as neuropeptide F (NPF), play key roles in flatworm physiology. Although progress has been made in the characterization of FaRP pharmacology in helminths, much remains to be learned. Most studies on nematodes have been done with Ascaris suum because of its large size. However, thanks to the Caenorhabditis elegans genome project, we know most about the FaRP complement of this free‐living animal. That essentially all C. elegans FaRPs are active on at least one A. suum neuromuscular system argues for conservation of ligand‐receptor recognition features among the Nematoda. Structure‐activity studies on nematode FaRPs have revealed that structure‐activity relationship (SAR) “rules” differ considerably among the FaRPs. Second messenger studies, along with experiments on ionic dependence and anatomical requirements for activity, reveal that FaRPs act through many different mechanisms. Platyhelminth FaRPs are myoexcitatory, and no evidence exists of multiple FaRP receptors in flatworms. Interestingly, there are examples of cross‐phylum activity, with some nematode FaRPs being active on flatworm muscle. The extent to which other invertebrate FaRPs show cross‐phylum activity remains to be determined. How FaRPergic nerves contribute to the control of behavior in helminths, and are integrated with non‐neuropeptidergic systems, also remains to be elucidated.

Terminal-Nerve-Derived Neuropeptide Y Modulates Physiological Responses in the Olfactory Epithelium of Hungry Axolotls (Ambystoma mexicanum)

The vertebrate brain actively regulates incoming sensory information, effectively filtering input and focusing attention toward environmental stimuli that are most relevant to the animals behavioral context or physiological state. Such centrifugal modulation has been shown to play an important role in processing in the retina and cochlea, but has received relatively little attention in olfaction. The terminal nerve, a cranial nerve that extends underneath the lamina propria surrounding the olfactory epithelium, displays anatomical and neurochemical characteristics that suggest that it modulates activity in the olfactory epithelium. Using immunocytochemical techniques, we demonstrate that neuropeptide Y (NPY) is abundantly present in the terminal nerve in the axolotl (Ambystoma mexicanum), an aquatic salamander. Because NPY plays an important role in regulating appetite and hunger in many vertebrates, we investigated the possibility that NPY modulates activity in the olfactory epithelium in relation to the animals hunger level. We therefore characterized the full-length NPY gene from axolotls to enable synthesis of authentic axolotl NPY for use in electrophysiological experiments. We find that axolotl NPY modulates olfactory epithelial responses evoked by l-glutamic acid, a food-related odorant, but only in hungry animals. Similarly, whole-cell patch-clamp recordings demonstrate that bath application of axolotl NPY enhances the magnitude of a tetrodotoxin-sensitive inward current, but only in hungry animals. These results suggest that expression or activity of NPY receptors in the olfactory epithelium may change with hunger level, and that terminal nerve-derived peptides modulate activity in the olfactory epithelium in response to an animals changing behavioral and physiological circumstances.

Structure-activity relationships of KNEFIRFamide (AF1), a nematode FMRFamide-related peptide, on Ascaris suum muscle

Analogues of KNEFIRFamide (Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2; AF1), an FMRFamide-related peptide (FaRP) originally isolated from Ascaris suum, were characterized in an A. suum muscle tension assay. AF1 had biphasic effects on this preparation, inducing a brief relaxation followed by excitation and spastic paralysis. Activity of AF1 in this assay was eliminated by N-terminal deletions and by deamidation of the carboxy-terminus. The potency of AF1 was greatly reduced by alanine substitution for any residue. Peptides that retained activity did not show the biphasic response observed with AF1, suggesting that the inhibitory and excitatory phases seen with AF1 may be due to activation of distinct receptors. The basis for the marked differences in potency observed between AF1 and the structurally related nematode FaRP, AF2 (KHEYLRFamide) was also tested. AF2 is approximately 1000-fold more potent than AF1 in this assay, but has physiological effects that are otherwise indistinguishable. KNEYIRFamide and KNEFLRFamide induced characteristic AF1/AF2 responses, but were much less potent than the native peptides. In contrast, KHEYIRFamide resembled AF1 in potency and pattern of responses. These data suggest that AF1 and AF2 act at distinct receptors, and hypothesis supported by the observation that KNEFIAFamide antagonized the effects of AF1 but not of AF2.

Discovery of multiple neuropeptide families in the phylum Platyhelminthes.

Available evidence shows that short amidated neuropeptides are widespread and have important functions within the nervous systems of all flatworms (phylum Platyhelminthes) examined, and could therefore represent a starting point for new lead drug compounds with which to combat parasitic helminth infections. However, only a handful of these peptides have been characterised, the rigorous exploration of the flatworm peptide signalling repertoire having been hindered by the dearth of flatworm genomic data. Through searches of both expressed sequence tags and genomic resources using the basic local alignment search tool (BLAST), we describe 96 neuropeptides on 60 precursors from 10 flatworm species. Most of these (51 predicted peptides on 14 precursors) are novel and are apparently restricted to flatworms; the remainder comprise nine recognised peptide families including FMRFamide-like (FLPs), neuropeptide F (NPF)-like, myomodulin-like, buccalin-like and neuropeptide FF (NPFF)-like peptides; notably, the latter have only previously been reported in vertebrates. Selected peptides were localised immunocytochemically to the Schistosoma mansoni nervous system. We also describe several novel flatworm NPFs with structural features characteristic of the vertebrate neuropeptide Y (NPY) superfamily, previously unreported characteristics which support the common ancestry of flatworm NPFs with the NPY-superfamily. Our dataset provides a springboard for investigation of the functional biology and therapeutic potential of neuropeptides in flatworms, simultaneously launching flatworm neurobiology into the post-genomic era.

An eye on RNAi in nematode parasites

RNA interference (RNAi) has revolutionised approaches to gene function determination. From a parasitology perspective, gene function studies have the added dimension of providing validation data, increasingly deemed essential to the initial phases of drug target selection, pre-screen development. Notionally advantageous to those working on nematode parasites is the fact that Caenorhabditis elegans research spawned RNAi discovery and continues to seed our understanding of its fundamentals. Unfortunately, RNAi data for nematode parasites illustrate variable and inconsistent susceptibilities which undermine confidence and exploitation. Now well-ensconced in an era of nematode parasite genomics, we can begin to unscramble this variation.

Neuropeptide-like protein diversity in phylum Nematoda

This study reports the identification of nematode neuropeptide-like protein (nlp) sequelogs from the GenBank expressed sequence tag (EST) database, using BLAST (Basic Local Alignment Search Tool) search methodology. Search strings derived from peptides encoded by the 45 known Caenorhabditis elegans nlp genes were used to identify more than 1000 ESTs encoding a total of 26 multi-species nlp sequelogs. The remaining 18 nlps (nlp-4, -16, -24 through -36, -39, -41 and -45) were identified only in C. elegans, while the sole EST representative of nlp-23 was from Caenorhabditis remanei. Several ESTs encoding putative antibacterial peptides similar to those encoded by the C. elegans genes nlp-24-33 were observed in several parasite species. A novel gene (nlp-46) was identified, encoding a single, amidated dodecapeptide (NIA[I/T]GR[G/A]DG[F/L]RPG) in eight species. Secretory signal peptides were identified in at least one species representing each nlp sequelog, confirming that all 46 nematode nlp genes encode secretory peptides. A random sub-set of C. elegans NLPs was tested physiologically in Ascaris suum ovijector and body wall muscle bioassays. None of the peptides tested were able to modulate ovijector activity, while only three displayed measurable myoactivity on somatic body wall muscle. AFAAGWNRamide (from nlp-23) and AVNPFLDSIamide (nlp-3) both produced a relaxation of body wall muscle, while AIPFNGGMYamide (nlp-10) induced a transient contraction. Numerical analyses of nlp-encoding ESTs demonstrate that nlp-3, -13, -14, -15 and -18 are amongst the most highly represented transcripts in the dataset. Using available bioinformatics resources, this study delineates the nlp complement of phylum Nematoda, providing a rich source of neuropeptide ligands for deorphanisation of nematode neuropeptide receptors.